Light-regulated translocation of signaling proteins in Drosophila photoreceptors.

Illumination of Drosophila photoreceptor cells induces multi-facet responses, which include generation of the photoreceptor potential, screening pigment migration and translocation of signaling proteins which is the focus of recent extensive research. Translocation of three signaling molecules is covered in this review: (1) Light-dependent translocation of arrestin from the cytosol to the signaling membrane, the rhabdomere, determines the lifetime of activated rhodopsin. Arrestin translocates in PIP3 and NINAC myosin III dependent manner, and specific mutations which disrupt the interaction between arrestin and PIP3 or NINAC also impair the light-dependent translocation of arrestin and the termination of the response to light. (2) Activation of Drosophila visual G protein, DGq, causes a massive and reversible, translocation of the alpha subunit from the signaling membrane to the cytosol, accompanied by activity-dependent architectural changes. Analysis of the translocation and the recovery kinetics of DGq(alpha) in wild-type flies and specific visual mutants indicated that DGq(alpha) is necessary but not sufficient for the architectural changes. (3) The TRP-like (TRPL) but not TRP channels translocate in a light-dependent manner between the rhabdomere and the cell body. As a physiological consequence of this light-dependent modulation of the TRP/TRPL ratio, the photoreceptors of dark-adapted flies operate at a wider dynamic range, which allows the photoreceptors enriched with TRPL to function better in darkness and dim background illumination. Altogether, signal-dependent movement of signaling proteins plays a major role in the maintenance and function of photoreceptor cells.     

An eGFP-based genetic screen for defects in light-triggered subcelluar translocation of the Drosophila photoreceptor channel TRPL

Signaling at the plasma membrane is modulated by up- and downregulation of signaling proteins. A prominent example for this type of regulation is the Drosophila TRPL ion channel that changes its spatial distribution within the photoreceptor cell. In dark-raised flies TRPL is localized in the rhabdomeral photoreceptor membrane and it translocates to the cell body upon illumination. It has been shown that TRPL translocation depends on the activation of the phototransduction cascade and requires the presence of functional rhodopsin as well as Ca2+-influx through a second lightactivated ion channel, TRP. However, little is known about the cell biological mechanism underlying TRPL translocation. Here we describe a FRT/FLP screen designed to isolate mutants defective in TRPL internalization based on the localization of eGFP-tagged TRPL in the eyes of living flies. We mutated chromosome arms 2L, 2R and 3R and isolated 12 mutants that failed to internalize TRPL. We found that four mutants did not complement genes known to affect TRPL translocation, which are trp, ninaE and inaD. Two of the isolated mutants represent new alleles of trp and ninaE. The trp allele contains a premature stop codon after amino acid 884, whereas the ninaE allele has a mutation resulting in the substitution P193S. As determined biochemically no TRP or rhodopsin protein, respectively, was expressed in the eyes of these mutants. The absence of TRP or rhodopsin in the isolated mutants readily explains the defect in TRPL internalization and proves the feasibility of our genetic screen.     

Translocation of the Drosophila transient receptor potential-like (TRPL) channel requires both the N- and C-terminal regions together with sustained Ca2+ entry

In Drosophila photoreceptors the transient receptor potential-like (TRPL), but not the TRP channels undergo light-dependent translocation between the rhabdomere and cell body. Here we studied which of the TRPL channel segments are essential for translocation and why the TRP channels are required for inducing TRPL translocation. We generated transgenic flies expressing chimeric TRP and TRPL proteins that formed functional light-activated channels. Translocation was induced only in chimera containing both the N- and C-terminal segments of TRPL. Using an inactive trp mutation and overexpressing the Na(+)/Ca(2+) exchanger revealed that the essential function of the TRP channels in TRPL translocation is to enhance Ca(2+)-influx. These results indicate that motifs present at both the N and C termini as well as sustained Ca(2+) entry are required for proper channel translocation.     

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.     

The Drosophila TRPL ion channel shares a Rab-dependent translocation pathway with rhodopsin

The Drosophila visual transduction cascade is embedded in the rhabdomeres of photoreceptor cells and culminates in the opening of the two ion channels, TRP and TRPL. TRPL translocates from the rhabdomeres to the cell body upon illumination and vice versa when flies are kept in the dark. Here, we studied the mechanisms underlying the light-dependent internalization of TRPL. Co-localization of TRPL and rhodopsin in endocytic particles revealed that TRPL is internalized by a vesicular transport pathway that is also utilized, at least partially, for rhodopsin endocytosis. TRPL internalization is attenuated under light conditions that result in a high rate of rhodopsin internalization and is highest in orange light that result in very little rhodopsin internalization. In line with a canonical vesicular transport pathway, we found that rab proteins, Rab5 and RabX4, are required for the internalization of TRPL into the cell body. Our results provide insight into stimulus-dependent internalization of a prominent member of the TRP superfamily.     

Mechanisms underlying stage-1 TRPL channel translocation in Drosophila photoreceptors

Background

TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels.

Methodology/Principal Findings

We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content.

Conclusions/Significance

Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a light-dependent anchor in the rhabdomere.     

Chemical excitation and inactivation in photoreceptors of the fly mutants trp and nss

The Drosophila and Lucilia photoreceptor mutants, trp and nss, respond like wild-type flies to a short pulse of intense light or prolonged dim light; however, upon continuous intense illumination, the trp and nss mutants are unable to maintain persistent excitation. This defect manifests itself by a decline of the receptor potential toward baseline during prolonged intense illumination with little change in the shape or amplitude of the quantal responses to single photons (quantum bumps). Previous work on the trp and nss mutants suggests that a negative feedback loop may control the rate of bump production. Chemical agents affecting different steps of the phototransduction cascade were used in conjunction with light to identify a possible branching point of the feedback loop and molecular stages which are affected by the mutation. Fluoride ions, which in the dark both excite and adapt the photoreceptors of wild-type flies, neither excite nor adapt the photoreceptors of the trp and nss mutants. The hydrolysis-resistant analogue, GTP gamma S, which excites the photoreceptors of wild-type flies, resulting in noisy depolarization, markedly reduces the light response of both mutant flies. Intracellular recordings revealed, however, that the inhibitory effect of GTP gamma S on the nss mutant was accompanied neither by any significant depolarization nor by an increase in the noise, and thus was very different from the effect of a dim background light. The combination of inositol trisphosphate and diphosphoglycerate (InsP3 + DPG), which efficiently excites the photoreceptors of wild-type Lucilia, also excites the photoreceptors of nss Lucilia mutant. The InsP3 + DPG together act synergistically with light to accelerate the decline of the response to light in the mutant flies. These results suggest that the fly phototransduction pathway involves a feedback regulatory loop, which branches subsequent to InsP3 production and regulates guanine nucleotide-binding protein (G protein)-phospholipase C activity. A defect in this regulatory loop, which may cause an unusually low level of intracellular Ca2+, severely reduces the triggering of bumps in the mutants during intense prolonged illumination.     

TRPgamma, a drosophila TRP-related subunit, forms a regulated cation channel with TRPL

TRP and TRPL are two light-sensitive cation channel subunits required for the Drosophila photoresponse; however, our understanding of the identities, subunit composition, and function of the light-responsive channels is incomplete. To explain the residual photoresponse that remains in the trp mutant, a third TRP-related subunit has previously been proposed to function with TRPL. Here, we identify such a subunit, TRPgamma. We show that TRPgamma is highly enriched in photoreceptor cells and preferentially heteromultimerizes with TRPL in vitro and in vivo. The N-terminal domain of TRPgamma dominantly suppressed the TRPL-dependent photoresponse, indicating that TRPgamma-TRPL heteromultimers contribute to the photoresponse. While TRPL and TRPgamma homomultimers are constitutively active, we demonstrate that TRPL-TRPgamma heteromultimers form a regulated phospholipase C- (PLC-) stimulated channel.     

Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P₂ in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P₂ was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P₂ is not an inhibitor of TRPL channel activation. PI(4,5)P₂ hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P₂ levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P₂ is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.     

Open channel block by Ca2+ underlies the voltage dependence of drosophila TRPL channel

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca(2+) blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca(2+) block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca(2+) that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca(2+) affinity are active.     

Short-term exposure to dim light at night disrupts rhythmic behaviors and causes neurodegeneration in fly models of tauopathy and Alzheimer's disease

The accumulation and aggregation of phosphorylated tau proteins in the brain are the hallmarks for the onset of Alzheimer's disease (AD). In addition, disruptions in circadian rhythms (CRs) with altered sleep-wake cycles, dysregulation of locomotion, and increased memory defects have been reported in patients with AD. Drosophila flies that have an overexpression of human tau protein in neurons exhibit most of the symptoms of human patients with AD, including locomotion defects and neurodegeneration. Using the fly model for tauopathy/AD, we investigated the effects of an exposure to dim light at night on AD symptoms. We used a light intensity of 10 lux, which is considered the lower limit of light pollution in many countries. After the tauopathy flies were exposed to the dim light at night for 3 days, the flies showed disrupted CRs, altered sleep-wake cycles due to increased pTau proteins and neurodegeneration, in the brains of the AD flies. The results indicate that the nighttime exposure of tauopathy/AD model Drosophila flies to dim light disrupted CR and sleep-wake behavior and promoted neurodegeneration.     

The activity of the TRP-like channel depends on its expression system

The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation.     

The activity of the TRP-like channel depends on its expression system

The Drosophila light activated TRP and TRPL channels have been a model for TRPC channel gating. Several gating mechanisms have been proposed following experiments conducted on photoreceptor and tissue cultured cells. However, conclusive evidence for any mechanism is still lacking. Here, we show that the Drosophila TRPL channel expressed in tissue cultured cells is constitutively active in S2 cells but is silent in HEK cells. Modulations of TRPL channel activity in different expression system by pharmacology or specific enzymes, which change the lipid content of the plasma membrane, resulted in conflicting effects. These findings demonstrate the difficulty in elucidating TRPC gating, as channel behavior is expression system dependent. However, clues on the gating mechanism may arise from understanding how different expression systems affect TRPC channel activation.     

Serotonin modulates a photic response in circadian locomotor rhythmicity of adults of the blow fly, Calliphora vicina

Abstract. The present experiments were undertaken to explore a role for serotonin (5-hydroxytryptamine, 5-HT) in modulating photic signal transduction in photoreceptors of the blow fly, Calliphora vicina. Injection of p-chlorophenylalanine (pCPA) into the haemolymph appeared to reduce sensitivity to the photic effects of constant ‘bright’ light (LL hyperactivity and circadian arrhythmicity). After drug injection in bright LL, flies continued with a free-running rhythm as in constant darkness (DD) or with a lengthened period τ as in ‘dim’ LL. When 5-HT was injected into flies kept in dim LL, they became hyperactive and arrhythmic as in bright LL. This finding suggests a potential role for serotonin as mediator of circadian changes in the insect visual system including extraretinal photoreceptors.     

Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.     

Regulation of Drosophila TRPC channels by protein and lipid interactions

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, proteins likely to be important components of calcium influx pathways. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PI(4,5)P(2) hydrolysis through a mechanism in which IP(3) receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that the accurate turnover of PI(4,5)P(2) generated lipid messengers in essential for regulating TRP and TRPL activity. TRP channels also appear to exist in the context of a macromolecular complex containing key components involved in activation such as phospholipase Cbeta and protein kinase C. This complex may be important for activation. The role of these protein and lipid elements in regulating TRP and TRPL activity is discussed in this review.     

TRP and calcium stores in Drosophila phototransduction.

Phototransduction in Drosophila is mediated by the ubiquitous phosphoinositide cascade, leading to opening of the TRP and TRPL channels, which are prototypical members of a novel class of membrane proteins. Drosophila mutants lacking the TRP protein display a response to light that declines to the dark level during illumination. It has recently been suggested that this response inactivation results from a negative feedback by calcium-calmodulin, leading to closure of the TRPL channels. It is also suggested that in contrast to other phosphoinositide-mediated systems, Ca2+ release from internal stores is neither involved in channel activation nor in phototransduction in general. We now show that inactivation of the light response in trp photoreceptors is enhanced upon reduction of the intracellular Ca2+ concentration. Furthermore, in Ca(2+)-free medium, when there is no Ca2+ influx into the photoreceptors, we demonstrate a significant elevation of intracellular Ca2+ upon illumination. This elevation correlates with ability of the cells to respond to light. Accordingly, malfunctioning of Ca2+ stores, either by Ca2+ deprivation or by application of the Ca2+ pump inhibitor, thapsigargin, confers a trp phenotype on wild type flies. The results indicate that the response inactivation in trp cells results from Ca2+ deficiency rather than from Ca(2+)-dependent negative feedback. The results also indicate that there is light-induced release of Ca2+ from intracellular stores. Furthermore, the response to light is correlated to Ca2+ release, and normal function of the stores is required for prolonged excitation. We suggest that phototransduction in Drosophila depends on Ca(2+)-release mediated signalling and that TRP is essential for the normal function of this process.     

Regulation of Drosophila TRPC channels by lipid messengers

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, which are important components of calcium influx pathways in virtually all cells. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PIP₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that one or more PIP₂ generated lipid messengers, as well as PIP₂ itself, are essential for regulating TRP and TRPL activity. Evidence on the role of these lipid elements in regulating TRP and TRPL activity is discussed in this review.     

Linoleic acid inhibits TRP channels with intrinsic voltage sensitivity: Implications on the mechanism of linoleic acid action

Open channel block (OCB) is a process by which ions bind to the inside of a channel pore and block the flow of ions through that channel. Repulsion of the blocking ions by membrane depolarization is a known mechanism for open channel block removal. For the N-methyl-D-aspartate (NMDA) channel, this mechanism is necessary for channel activation and is involved in neuronal plasticity. Several types of Transient Receptor Potential (TRP) channels, including the Drosophila TRP and TRP-Like (TRPL) channels, also exhibit open channel block. For the Drosophila TRP and TRPL channels, removal of open channel block is necessary for the production of the physiological response to light. Recently, we have shown that lipids such as polyunsaturated fatty acids (PUFAs), represented by linoleic acid (LA), alleviate OCB under physiological conditions, from the Drosophila TRP and TRPL channels and from the mammalian NMDA channel. Here we show that OCB removal by LA is not confined to the Drosophila TRPs but also applies to mammalian TRPs such as the heat activated TRPV3 channel. TRPV3 shows OCB alleviation by LA, although it shares little amino acid sequence homology with the Drosophila TRPs. Strikingly, LA inhibits the heat-activated TRPV1 and the cold temperature-activated TRPM8 channels, which are intrinsic voltage sensitive channels and do not show OCB. Together, our findings further support the notion that lipids do not act as second messengers by direct binding to a specific site of the channels but rather act indirectly by affecting the channel-plasma membrane interface.     

The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk

The light preferences of fruit flies were tested by 2 different means. First, flies were allowed to choose between different illuminations, and their favorite resting, grooming, and feeding places were determined with an infrared-sensitive camera. Second, the activity levels of the animals during their main daily activity period were determined photoelectrically (via infrared light beams) under different light intensities. Both methods revealed that the flies prefer dim light. They rested, groomed, and fed preferentially in places with a light intensity between 5 and 10 lux, and they showed the highest activity level when the light intensity during the day was kept at 10 lux. Furthermore, when dawn and dusk were simulated by logarithmically increasing/decreasing the light intensity during a 1.5-h interval, the flies' activity maxima occurred at about 7.5 lux during early dawn and late dusk. The results suggest that fruit flies time their clocks by early dawn and late dusk and avoid bright light during the day.